Tumour therapy is mainly implemented by means of the use of substances aimed at killing the tumour cells. This can be achieved with cytotoxic substances which have to enter the tumour cell in order to exert their full effect, or by means of treatment of the tumour cells with radiation with sufficient energy to kill the cell. In both cases, there is the problem of delivering the substance as selectively as possible to the target cell, so as to avoid possible damage to the surrounding healthy cells. In the case of radiopharmaceuticals, i.e. of substances bearing radioactive portions, the problem of selectively delivering the active part (that is to say, the radioactive portion) to the tumour target, avoiding the spread of radionuclide in the body or in the healthy cells surrounding the tumour, is of particular concern.
One particularly effective method for tumour detection and therapy is described in patent EP 0 496 074. The protocol of this patent has been applied to the so-called Pretargeted Antibody-Guided Radioimmunotherapy (PAGRIT) of brain tumours. In this method, avidin is injected into the human subject, after the biotinylated anti-tenascin monoclonal antibody (Mab-B), to remove any free Mab-B, not bound to the tumour, from the bloodstream by forming complexes with it that are effectively eliminated by the liver (chase effect). An infusion of streptavidin is then administered for the purposes of obtaining better avidination of the tumour compared to that obtainable with avidin, whose permanence in the blood is too short compared to that of streptavidin.
Though the system has shown positive clinical responses (Cremonesi M., et al., 1999; Paganelli G., et al., 1999; Paganelli G., et al., 2001), one major limiting factor consists in the strong immune response caused by streptavidin (Paganelli G., et al., 1997). For the purposes of overcoming these two obstacles, i.e. the high degree of immunogenicity of streptavidin and the rapid clearance of avidin, avidins have been used which are chemically modified by covalently binding polyoxyethylene glycol (PEG) chains to avidin, with various levels of derivatisation based on the use of straight or branched PEGs of different molecular weights. Preliminary studies have revealed that, with the increase in the degree of functionalisation of avidin with PEG (hereinafter referred to as pegilation), there is an increase in the plasma half-life of avidin, a reduction in immunogenicity, and an improvement in the specific biodistribution of the substance in relation to the tumour.
Since the ability of avidin-PEG to bind to Mab-B biotin is reduced by pegilation, the result is a reduction in the potency of the derivatives (Chinol M. et al., 1998).
A solution to this problem has been proposed in patent application WO 94/23759, filed in the name of Immunomedics, where avidin multipolymers are described based on the chemical derivatisation of high-molecular-weight molecules, preferably greater than 5,000 Da, such as dextrane, proteins, and polycarboxylic acids. But none of the five multimers effectively described in the patent has been characterised in terms of its potency of action in the pretargeting procedure or in other procedures.
As demonstrated in the invention described herein, the general concept of multimerisation (also including dimerisation), given in the above-mentioned patent application WO 94/237599, fails to provide complete and sufficient instructions for the average technician in finding a generic avidin multimer capable of fulfilling the necessary requirements in the application of the three-step pretargeting method. In fact, different diavidins, obtained using different bifunctional cross-linkers, though possessing the same ability to bind free biotin, differ in their potency when assayed in vitro in three-step pretargeting, to the extent that, in certain cases, they prove to be completely inefficacious.
This observation indicates that the multimerisation of avidin does not automatically produce useful functional products, but that biological characterisation is necessary for the choice of a potentiated molecule suitable for pretargeting.